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Avian influenza virus np protein antigen epitope polypeptide and colloidal gold test kit

A bird flu virus and antigen epitope technology, applied in peptides, instruments, analytical materials, etc., can solve the problems that it is difficult to understand the real law of changes, surface proteins are prone to mutation, etc.

Active Publication Date: 2019-06-18
青岛蔚蓝动物保健集团有限公司 +3
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The main reason why the epidemic of avian influenza has not been effectively controlled so far is that its surface protein is extremely prone to mutation, making it difficult for people to understand the real law of its change

Method used

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  • Avian influenza virus np protein antigen epitope polypeptide and colloidal gold test kit
  • Avian influenza virus np protein antigen epitope polypeptide and colloidal gold test kit
  • Avian influenza virus np protein antigen epitope polypeptide and colloidal gold test kit

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0030] Example 1 Identification of linear epitope of avian influenza H9N2 subtype virus

[0031] 1 Materials and methods

[0032] Main experimental materials and experimental animals: SP2 / 0 cells are stored in the applicant's laboratory; recombinant plasmid Pet32a-NP expressing AIV (H9N2) NP protein is constructed by our laboratory; AIV (H9N2) virus is stored in our laboratory; SPF chicken embryo Purchased from Merial; SPF BALB / c female mice aged 6-8 weeks were purchased from the Experimental Animal Center of Beijing Medical College.

[0033] Main reagents: PEG6000 (Merck), sucrose (Shanghai Sinopharm), Freund's complete adjuvant and Freund's incomplete adjuvant (SIGMA), Rabbit anti mouse-HRP (SIGMA), Rabbit anti mouse-FITC (SIGMA), phage display Peptide Library Kit (NEB)

[0034] Purification and identification of recombinant antigens: The applicant screened avian influenza viruses from sick individuals who had been injected with avian influenza virus vaccines but still developed av...

Embodiment 2

[0053] Example 2 Optimization of monoclonal antibody epitope

[0054] 1. Purify the monoclonal antibodies produced by the two cell lines of NP-Mab 1B2 and 2B5 separately. The purified monoclonal antibodies are tested for HI. The test results show that 1B2 has a higher HI titer than 2B5.

[0055] Table 3. NP-Mab antibody titer detection

[0056]

[0057] 2. Determination of relative affinity constant of monoclonal antibody

[0058] The thiocyanate elution method is used to determine the relative affinity constant of monoclonal antibodies. The specific steps are as follows:

[0059] (1) Take the purified H9 subtype avian influenza virus as an antigen-coated and blocked indirect ELISA plate, dilute the monoclonal antibody to be tested to a saturated concentration, add 100 μL to each well, and incubate at 37°C for 1 hour;

[0060] (2) After washing 3 times with PBST, different concentrations of thiocyanate are eluted, and NaSCN solutions with concentrations of 0, 0.5, 1.0, 1.5, 2.0, 2.5, 3.0...

Embodiment 3H9

[0066] Example 3H9 subtype avian influenza virus colloidal gold test strip

[0067] 1. Preparation of monoclonal antibodies against H9 subtype avian influenza virus outer membrane hemagglutinin antigen and rabbit anti-mouse IgG antibodies

[0068] Preparation of monoclonal antibodies against H9 subtype avian influenza virus nucleoprotein antigen: (Hybridoma cells are prepared and preserved by our laboratory) The selected hybridoma cells are resuscitated and then expanded and cultured to prepare ascites, namely H9 subtype avian influenza virus antibodies. ELISA to identify the activity and titer of the monoclonal antibody against the H9 subtype avian influenza virus nucleoprotein antigen, and purify it for use;

[0069] Preparation of rabbit anti-mouse IgG antibody: use conventional methods to immunize rabbits with purified mouse IgG as the immune antigen, collect venous blood two weeks later, measure the activity and titer of anti-mouse IgG antibody by ELISA, and purify it for use.

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Abstract

The invention relates to an avian influenza virus NP proteantigen epitope peptide and a colloidal gold test paper box thereof. The invention provides an avian influenza H9N2 subtype virus NP protein linear epitope peptide, and the antigen epitope peptide has the amino acid sequence of SEQ ID NO: 1. The antigen epitope peptide provided by the invention is used for preparing a product for detecting avian influenza virus. The avian influenza virus NP protein B cellular antigen epitope peptide is identified for providing a ground for further establishing a method of efficiently detecting avian influenza, and also lays a foundation for studying the structure and function of NP protein.

Description

Technical field [0001] The invention belongs to the technical field of animal medicine and biotechnology, and particularly relates to an avian influenza virus NP protein antigen epitope polypeptide and a colloidal gold test box prepared therefrom. Background technique [0002] The H9N2 subtype avian influenza virus (AIV) is an influenza A virus that causes a variety of symptoms in poultry from the respiratory system to severe systemic sepsis. It belongs to the influenza A virus. In 1966, the first H9N2 subtype avian influenza virus was isolated by HoMee from a turkey with mild respiratory disease. It was not until 1994 that my country first reported the isolation of the H9N2 subtype of avian influenza, and related reports appeared one after another. However, since the pathogenicity and mortality of the H9N2 subtype avian influenza virus are not as high as those of H5N1 and H7H1, the infection is often difficult to detect and does not attract the attention of farmers. In fact, H...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K7/06G01N33/569
CPCG01N33/558G01N33/56983G01N33/577
Inventor 刘晓婧蒋贻海凌红丽赵明丁江由佳李明举张志东王艳玲武利利周大卫王晓艺杨莉卢香玲王海燕
Owner 青岛蔚蓝动物保健集团有限公司